Distribution Of α-SMA Research Articles

Exploration of Perturbed Liver Fibrosis-Related Factors and Collagen Type I in Animal Model of Non-Alcoholic Fatty Liver Disease.

To determine their involvement in the onset of the disease, we investigated the changing levels of liver fibrosis-related proteins, namely, type-I collagen, α-smooth muscle actin (α-SMA), and transforming growth factor β1 and β3 (TGF-β1, β3). The four groups of Sprague-Dawley (SD) rats were involved in the study, namely, (i) normal control group, (ii) high-fat diet group (HFD), (iii) carbon tetrachloride (CCl4) group, and (iv) NAFLD group (animal model) which were chosen at random. The NAFLD model received HFD combined with subcutaneous injection of small doses of CCl4. Histopathological examination confirmed extent of liver fibrosis, while other immunological and molecular methods were used to evaluate expression and distribution of α-SMA, type I collagen TGF-β1 and TGF-β3, at both m-RNA and protein levels. In contrast to the normal control group, the NAFLD group showed moderately elevated expressions of TGF-β1, α-SMA, and type I collagen, which was proportional on temporal scale of NAFLD persistence in the model (P < 0.05). In the early phage of NAFLD, enhancement in the mRNA transcripts and, henceforth, protein expression of TGF-β3 was observed. However, these were found to be downregulated in case of liver fibrosis (P < 0.05). This NAFLD rat model shows the histopathologic changes of human NAFLD and is suitable for the study of NAFLD pathogenesis. These findings suggest that type I collagen and the liver fibrosis-related factors TGF- β1, TGF- β3, and α-SMA may be significant contributors to NAFLD. Although NAFLD model is previously demonstrated by other researchers, our study is novel in terms of exploration of involvement of fibrosis-related factors and in particular aforementioned proteins at the early stage of NAFLD vis-à-vis dynamics of type-I collagen distribution.

Investigating the potential for reversal of myofibroblast activation in human cardiac fibroblasts in 2D culture

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): BHF MRC Introduction Cardiac fibroblasts (cFbs) are responsible for deposition of extracellular matrix in the heart, providing support to the contracting myocardium and contributing to a myriad of physiological signalling processes. Prolonged and excessive activation of cFbs, via stimulation by transforming growth factor β (TGF-β), causes conversion of cFbs into myofibroblasts. Myofibroblasts are believed to cause pathological cardiac remodelling and to contribute to heart failure and arrhythmias. Reversion of myofibroblasts into cFbs has been demonstrated in rodent cells; it has yet to be explored in human cells. Purpose To characterise the effects of long-term 2D standard culture on the activation status of human cFbs. To identify the potential for human myofibroblasts to dedifferentiate back to cFbs. Methods Primary human cFbs were cultured in Corning Costar flasks (Young’s modulus E = ∼3GPa) for up to 10 passages. Cells were subsequently plated onto dishes with a Young’s modulus of ∼3GPa, 25kPa and 2kPa in the presence or absence of TGF-β (10ng/ml) and/or TGF-β receptor I inhibitor SD208 (10nM) for up to 4 days. The proliferative capacity of the cells was assessed using the CyQUANT NF® assay. Cells were assessed for mRNA and protein expression of myofibroblast activation markers α-smooth muscle actin (α-SMA) and collagen-1 by qPCR and western blotting. The localised distribution of α-SMA was assessed by confocal microscopy. Results Human cardiac fibroblasts robustly expressed α-SMA. Proliferation was significantly decreased at 2kPa compared to higher Young’s moduli (mean percentage change over 2 days: 2kPa = 115.1, 25kPa = 191.4, 3GPa = 205.9, p &amp;lt; 0.0001). qPCR analysis revealed no significant changes in expression of myofibroblast gene markers α-SMA and collagen 1 at either ∼3GPa, 25kPa or 2kPa Young’s Moduli in the presence or absence of TGF-β treatment (median fold change (interquartile range [IQR]) versus control: TGF-β(α-SMA, 3GPa) = 1.226 (0.820); TGF-β(Collagen 1, 3GPa) = 1.636 (1.403); TGF-β(α-SMA, 25kPa) = 1.069 (7.030); TGF-β(Collagen 1, 25kPa) = 1.103 (0.411); TGF-β(α-SMA, 2kPa) = 0.800 (5.021); TGF-β(Collagen 1, 2kPa) = 1.629 (7.092); n = 2-3). These data was confirmed by western blotting (median relative protein expression (IQR) versus control: TGF-β(α-SMA, 3GPa) = 1.012 (0.500); TGF-β(Collagen 1, 3GPa) = 1.008 (1.466); TGF-β(α-SMA, 25kPa) = 1.321 (2.282); TGF-β(Collagen 1, 25kPa) = 0.944 (1.125); TGF-β(α-SMA, 2kPa) = 1.142 (0.705); TGF-β(Collagen 1, 2kPa) = 0.283 (1.127), p &amp;gt; 0.05; n = 2-3). TGF-β or SD208 treatment did not affect α-SMA expression when assessed by confocal microscopy. Conclusions Long-term culture of human cFbs in 2D format leads to a robust and persistent activation of myofibroblasts that is unresponsive to TGF-ß activation or inhibition. Ongoing work is focussed on investigating whether human myofibroblast de-differentiation is possible.

Upregulation of Protease-Activated Receptor 2 Promotes Proliferation and Migration of Human Vascular Smooth Muscle Cells (VSMCs).

BackgroundProtease-Activated Receptor 2 (PAR2), a G-protein-coupled receptor, has been proved to be enhanced in human coronary atherosclerosis lesions. We aimed to investigate whether PAR2 actively participates in the atherosclerosis process.Material/MethodsPAR2 expression was assessed in blood samples by RT-qPCR from healthy controls and patients with atherosclerosis. Human vascular smooth muscle cells (VSMCs) were treated with oxidative low-density lipoprotein (ox-LDL). After PAR2 overexpression by transfection, cell proliferation was determined by CCK-8, and cell migration was evaluated by Transwell assay. The protein expressions associated with cell growth and migration were measured by Western blot. The distribution of α-SMA in VSMCs was evaluated by immunofluorescence.ResultsExpression of PAR2 was higher in patients with atherosclerosis compared with normal controls. PAR2 mRNA and protein expression was increased in ox-LDL-treated VSMCs compared with control cells. Induced overexpression of PAR2 in VSMCs led to a reduction in α-SMA expression compared to controls. In addition, PAR2 overexpression caused increased migration compared to normal controls, and upregulated MMP9 and MMP14 expression. PAR-2 overexpression promoted cell proliferation compared to control cells, and increased expression levels of CDK2, and CyclinE1, but reduced levels of p27. We preliminary explored the potential mechanism of PAR2, and results showed that overexpression of PAR2 increased expression levels of VEGFA and Angiopoietin 2 compared to controls. Moreover, overexpression of PAR2 enhanced production of tissue factor and IL-8 compared to normal controls.ConclusionsPAR2 promotes cell proliferation and disrupts the quiescent condition of VSMCs, which may be a potential therapeutic target for atherosclerosis.

Effects of transplanted adipose derived stem cells on the expressions of α-SMA and DCN in fibroblasts of hypertrophic scar tissues in rabbit ears.

To study the effects of transplanted adipose derived stem cells (ADSCs) on the expressions of α-smooth muscle actin (α-SMA) and decorin (DCN) in fibroblasts of hypertrophic scar tissues in rabbit ears. Twelve New Zealand white rabbits were selected; the normal subcutaneous adipose tissues in inguinal region were removed, ADSCs were extracted via enzyme digestion, cultured in Dulbecco's modified Eagle's medium (DMEM) and inoculated into the culture dish (3–5×104 cells/ml). After the rabbit ear hypertrophic scar model was established successfully, the fibroblasts of hypertrophic scar tissues in rabbit ears were separated and cultured using the mechanical method combined with enzyme digestion, and the ADSCs and scar fibroblasts were cultured in non-contact Transwell co-culture system for 21 days (experimental group); the corresponding scar fibroblasts were cultured in an ordinary 6-well plate without any treatment for 21 days (control group). The content of collagen I in fibroblasts was detected using the enzyme-linked immunosorbent assay (ELISA) kit, the mRNA expressions of α-SMA and DCN were detected via reverse transcription-polymerase chain reaction (RT-PCR), the protein expressions of α-SMA and DCN were detected via western blot analysis, and the expressions and distribution of α-SMA and DCN were detected via immunofluorescence assay. The results of ELISA showed that the content of collagen I in experimental group was decreased significantly (p<0.01). The results of RT-PCR and western blot analysis revealed that the mRNA and protein expression levels of α-SMA were significantly decreased (P<0.01, but those of DCN were significantly increased (p<0.01). Moreover, the results of immunofluorescence assay showed that the expression of α-SMA in experimental group was significantly decreased, while the expression of DCN was significantly increased. ADSCs can inhibit the mRNA and protein expressions of α-SMA and promote the mRNA and protein expressions of DCN in in vitro culture system, and they are expected to be used in the prevention and treatment of pathological scars.

Abstract 4371: RRx-001 modulates intratumor blood flow in SCCVII and U87 tumors

Abstract The tumor-specific provascular effects of the anti-cancer agent RRx-001, a novel nitrogen oxide donor and allosteric hemoglobin modifier in Phase 1 clinical trials were investigated using contrast enhanced ultrasound (CEUS) and whole tissue section quantitative immunohistological staining. Changes in tumor blood flow by CEUS in SCCVII tumor bearing mice following RRx-001 (3, 6, 12 mg/kg) were dose and time dependent, reaching maximum perfusion 6 h after treatment, returning to baseline within 72 h. Microregional effects of RRx-001 (15 mg/kg) on tumor blood flow and oxygenation by immunohistological staining were studied in mouse SCCVII and human U87 tumors. SCCVII tumors exhibited regions of intermittent perfusion exemplified by co-localization of vessels with the hypoxia marker pimonidazole commonly occurring throughout the tissue. Only 21% of vessels showed perfusion after a bolus dose of the perivascular stain DiOC7. This increased to 28% 12 h post RRx-001. Paradoxically, an increase in tissue oxygenation was not seen while modestly increased pimonidazole binding was observed. However, preliminary radiobiological assessment suggested tumors were radiosensitized when irradiated 10 min after 12 mg/kg RRx-001. In the U87 tumor a decrease in vessel perfusion at 90 min post RRx-001 administration was observed. Blood flow over large areas of treated tumors was completely shut down. These areas stained positive for CD31 and tissue did not appear necrotic. Analysis indicated 25% reduction in the fraction of perfused vessels and un-perfused tissue by area at 90 min returning to near normal levels at 12 h. There was no significant increase in the fraction of necrosis at 12 h suggesting that the large un-perfused regions seen at 90 min had recovered rather than become necrotic. No significant change in tumor hypoxia was seen at 90 min or 12 h. For both tumor types, RRx-001 appeared to cause the loss of perfusion in large regions of the tumor however, at the 12 h time point, both tumor types showed increase in vessel perfusion but no significant decrease in hypoxia. These data suggest a redistribution of blood flow in both tumor types while differences between the tumors were related to tumor architecture and distribution of α-SMA. RRx-001 shows promise for short-term blood flow redistribution in tumors with a pericyte-rich vasculature with contractile proteins such as α-SMA. Expression of α-SMA in tumor vasculature could therefore be useful for predicting tumor response to RRx-001. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4371. doi:1538-7445.AM2012-4371

Pokeweed antiviral protein down-regulates Wnt/β-catenin signalling to attenuate liver fibrogenesis in vitro and in vivo

Background and aim Pokeweed antiviral protein (PAP) has been regarded as a strong negative regulator of Wnt/β-catenin signalling, which promotes tissue fibrogenesis. Whether PAP inhibits hepatic fibrosis remains unknown. Here, the inhibitory effects of PAP on hepatic fibrosis induced by CCl 4 in rats were monitored. Methods Plasmid pXF3H-PAP was transduced into liver in vivo and hepatic stellate cell (HSC-T6) in vitro. Changes in liver pathology were examined by haematoxylin and eosin (H&E) and Masson's trichrome staining. Hepatic function and the levels of hyaluronic acid (HA) and laminin (LN) in serum and hydroxyproline (Hyp) in liver were measured. We also observed the expressions and distribution of α-SMA, TIMP-1, smad3 and β-catenin in HSCs and liver, together with the viability of HSC-T6 cells. Results PAP obviously attenuated the histological changes, decreased the content of Hyp in liver and improved liver function in rats ( P < 0.05). PAP significantly reduced the expressions and distribution of α-SMA and β-catenin in vivo (0.0125 ± 0.002 vs 0.0374 ± 0.003, 0.0135 ± 0.003 vs 0.0382 ± 0.004; P < 0.05, respectively) and in vitro as well as the cell viability ( P < 0.05). Conclusions Our results revealed that PAP attenuated liver fibrogenesis through down-regulating the Wnt/β-catenin pathway. It provides us an alternative new strategy for the treatment of hepatic fibrosis.

Effect of tension on collagen arrangement

Objective To study the effect of mechanical tension on collagen arrangement and illustrate the relationship between tissue architecture and tension properties. Methods Cell morphologies, orientation and collagen arrangement of fibroblasts cultured in three different types of collagen gels with variation of mechanical tension were observed by phase contrast photomicrographs, light microscopy and transmission electron microscopy. Expression and distribution of a-smooth muscle actin (α-SMA) were investigated by immunohistochemistry. Results Phase contrast photomicrographs, light microscopy and transmission electron microscopy showed high level of tension distributed anisotropically in the monolayer gels and the anchored collagen gels, with bipolar shape of the fibroblasts, obvious polarity, arrangement of exogenous collagen fibres parallel to the long axis of the fibroblasts, especially prominent in monolayer gels. Low level of tension distributed isotropically was observed in floating collagen gels, with stellate morphology and arrangement of exogenous collagen fibres in a reticular array. Immunofluorescence showed that fibroblasts expressed high level of α-SMA protein distributed along the long axis of fibroblasts in the monolayer gels and the anchored collagen gels, especially in former ones. In contrast, few expression of α-SMA protein was found in floating collagen gels. Cell morphologies and orientation, expression and distribution of α-SMA as well as collagen arrangement of fibroblasts in the monolayer gels and the anchored collagen gels were similar to those in granulation tissue, whereas floating collagen gels resembled normal dermis or remodelled tissues. Conclusions Tissue architecture or morphology of the dermis are corresponding to tension proporties. Different tissue architectures are closely correlated with particular tension proporties. Key words: Tensile strength; Fibroblasts; Collagen; Cytoskeleton

The change of myofibroblast distribution in hyperoxia induced bronchopulmonary dysplasia in newborn rats

Objective To investigate the expression of alpha-smooth muscle actin (α-SMA) and collagen Ⅰ in lung tissue of newborn rats with hyperoxia induced bronchopulmonary dysplasia(BPD) and to explore the relationship between the myofibroblast and BPD. Methods Neonatal rats were exposed to room air or 85% oxygen for 21 days. Six rats of both groups were sacrificed at day 1,3,7,14 and 21. The left lung tissues were fixed, the right lung tissues were stored at - 80 ℃. The pathologic changes of lung were observed under optical microscope. Distribution of α-SMA in the lung were determined by immonohistochemical method. The protein and mRNA levels of collagen Ⅰ were detected by ELISA and RT-PCR respectively. Results Prolonged exposure to hyperoxia could result in secondary septum decreasing, enlarging terminal air space and increased collagen deposition in lung tissue. In hyperoxia group,α-SMA expression increased significantly in smooth muscle,alveolar septa interstitium and the surface of alveolar on day 14 and 21. But in air group,α-SMA expressed mainly at the tip of the secondary septa except in smooth muscle. Collagen Ⅰ deposition increased with hyperoxia exposure time prolonged. There was positive correlation between the expression of α-SMA and collagen Ⅰ mRNA in the lung tissue of hyperoxia group. Conclusion The pathological changes of lung injury induced by hyperoxia exposure in newborn rats are consistent with BPD of newborn infant. Abnormality of myofibroblast distribution may play an important role in pathogenesis of BPD. Key words: Bronchopulmonary dysplasia ; Alpha-smooth muscle actin ; Myofibroblast ; Rat ;